Gibberellic acid (GA3) is the most important gibberellin, a class of diterpenoid acids that function as plant growth regulators (Jefferys, E. G., Adv. Appl. Biol. 13:283-316 (1970)). GA3 affects stem elongation, elimination of dormancy, flowering, sex expression, enzyme induction and leaf and fruit senescence. GA3 is a high-value, industrially-important, biochemical with various applications in agriculture (L. M. Pastrana et al., “Interactions affecting gibberellic acid production in solid-state culture: A factorial study”; Enzyme and Microbial Technology 17:784-790 (1995), citing to Kumar, P. K. R., and Lonsane, B. K., Appl. Microbiol. Biotechnol. 34:145-148 (1990)). However, its high cost has restricted its use to premium crops (Jefferys, E. G., Adv. Appl. Biol. 13:283-316 (1970)).
GA3 has been obtained by chemical synthesis, extraction from plants and by microbial fermentation. Stereospecific chemical synthesis involves multiple steps and the use of expensive reagents. To date, chemical synthesis is not economically competitive with fermentation techniques. Direct extraction of GAs from higher plants is not economically feasible due to extremely low concentration of GAs in plant tissue. A large number of bacteria, actinomycetes and yeast cultures have been reported to produce GA3 or GA3-like substances, but most do not produce GA3 at commercially-feasible levels. Today, GA3 is produced by fermentation, mostly from fungi.
Fermentation Methods
GA3 production in fungal culture includes liquid surface fermentation, submerged fermentation, and solid state fermentation.
Liquid Surface Fermentation (LSF)
Liquid surface fermentation (LSF), often referred to as surface fermentation, was employed in earlier years for the production of GA3 but suffered from low yield (e.g. 3.3-9.2 g of crude powdered GA3 from 130 liters of medium) problems with scalability, contamination and more, and was discontinued (P. K. R. Kumar and B. K. Lonsane, “Microbial production of Gibberellins: state of the art” in Advances in Applied Microbiology. 34: 29-140 (1989)).
Submerged Fermentation (SmF)
Currently, GA3 is largely produced by submerged fermentation (SmF) of the fungus Gibberella fujikuroi. The fungus known as Fusarium moniliforme is also used; F. moniliforme is the anamorph (asexual stage) of Gibberella fujikuroi. GA3 is also synthesized from several bacteria such as Azotobacter, and Azospirillium in culture medium and from wild strains of fungi such as Sphaceloma sp., Phaeosphaeria sp., and Neurospora sp.
The initial pH values generally employed by various workers are within the range 3.5-5.8, especially around pH 5.5. Id. pH was not usually controlled during fermentation and thus resulted in a final pH differing from the starting pH, with reports of the final pH of 3.9-5.2, 1.8-1.9, or even slight alkalinization. Id.
The effect of temperature on the production of GA3 is dependent on the strain employed. The optimal temperatures reported for the production of GA3 using G. fujikuroi or F. moniliforme include 25° C.; 28.5-29.5° C.; 30° C.; 34° C.; 29± 0.5° C.; 27° C.; and 28° C. (See Id.)
Different workers have used a variety of carbon sources for the production of GAs, as reported by different authors. A combination of readily and slowly metabolizable carbon sources gave a higher yield of GA3. Id. Use of molasses led to decreased but economically useful yields. Id. There was a 49% decrease in the yield of GAs with stearic acid as compared to sucrose. Use of dairy waste as carbon source resulted in 0.75 g GA3/liter in 12 days. The production was completely inhibited by 1 ppm geraniol due to total inhibition of cell growth. A direct proportional relationship has been shown between the initial concentration of nitrogen supplied in the medium and the rate of product formation as well as the amount of metabolite produced. Id. See also Jefferys, E. G., Adv. Appl. Biol. 13:283-316 (1970)
Trace elements such as Fe, Cu, Mn, Zn, Al and Ca are required in the fermentative production of GAs. Id. Trace elements may be expressly added in excess or in combination, as in Raulin-Thom medium, or sufficient trace elements may be present as impurities in other ingredients of the fermentation medium. Id.
Other growth factors such as vitamins or Yeast extract may improve yield; while other heavy metals or the use of lines steel or stainless steel fermentation tanks can decrease yield. Id.
Various processes for production of GA3 are described in U.S. Pat. Nos. 2,842,051, 2,865,812, 2,906,671, 2,906,673 and 3,021,261. U.S. Pat. No. 2,865,812 reported a yield of 630 mg/L in 664 hrs. Eleazar et al. (“Optimization of gibberellic acid production by immobilized Gibberella fujikuroi mycelium in fluidized bioreactors”, Journal of Biotechnology, 76, 147-155 (2000)) reported yields by submerged fermentation as high as 2.862 g/L.
Solid Substrate Fermentation (SSF)
SSF (Solid Substrate Fermentation) is defined as any fermentation process performed on a non-soluble material that acts both as physical support and source of nutrients, in the absence of free flowing liquid. No free-moving water is present, but there is enough moisture present for the growth and metabolism of the microorganism. The low moisture content means that fermentation can only be carried out by a limited number of microorganisms, mainly yeasts and fungi, although some bacteria have been used. Work on the production of GAs using SSF technique was initiated in the early 1980s and the initial studies gave variable yields. No information was available on the economics of the process until early 1987. Three cases were examined wherein the yield of GA3 under the SSF technique were 0.825, 1.05 and 1.54 g/kg of Dry Mouldy Bran. The yield of GA3 in the fed-batch SSF Process was 1.54 g/kg of Dry Mouldy Bran as compared to 1.05 g/kg of Dry Mouldy Bran in the batch SSF process. Id. One group reported a yield of 3 g/kg by SSF (Bandelier, S., Renaud, R., and Durand, A., “Production of Gibberellic acid by Fed-batch solid state fermentation in an aseptic pilot-scale reactor”, Process Biochemistry, 32:141-145 (1997)). B. Tudzynski (“Biosynthesis of gibberellins in Gibberella fujikuroi: biomolecular aspects”; Appl. Microbiol Biotechnol (1999) 52:298-310) reported that German Patent Number DD252000 also described processes for GA3 by submerged fermentation, and reported yields of 8 g/kg.
Art-Known Fermentation Techniques Remain Expensive
Despite advances in fermentation technology, the cost of production of GA3 has been a deterrent to its widespread use. Additional cost considerations include problems in the downstream processing and, given the potency of GA3 to plants, removal and disposal of contaminated wastewater.